Insulin-like growth factor 1 (IGF-1) participates in the maintenance of the malignant phenotype by enhancing proliferation and protecting cancer cells from apoptosis. Insulin receptor substrate 1 (IRS-1) is a key intracellular signaling molecule of the IGF-1 pathway that connects the IGF-1 receptor to its downstream signaling pathways. Constitutive activation of IRS-1 is a frequent event in human tumors including breast cancer. Thus, any negative regulator of IRS-1 and IRS-1-mediated IGF-1 signaling may have potential antitumor activity. Tumor necrosis factor alpha (TNF-alpha) exerts its anti-proliferative effect on breast cancer cells by impairing IGF-1 signaling. Our preliminary studies suggest that protein phosphatase 4 (PP4) interacts with and down-regulates IRS-1 and IRS-4 following TNF-alpha stimulation. In this application, we will test our hypothesis that PP4 plays a role in mediating the antagonistic effect of TNF-alpha on IGF-1 signaling by dephosphorylating and down-regulating IRS-1, and thus exerts its tumor suppressive function in breast cancer cells. The specific aims are: Aim 1. Study the mechanism of IRS-1 dephosphorylation and down-regulation by PP4 in breast cancer cells. We will study the functional significance of IRS1-PP4 interaction in MCF-7 breast cancer cells. We will map the PP4 dephosphorylation site(s) within IRS-1 by 2-D phosphopeptide mapping and microsequencing. We will determine the functional significance of PP4-mediated dephosphorylation by studying phosphorylation-deficient and phosphorylation-mimetic mutants of IRS-1 in IGF-1 signaling in breast cancer cells. Aim 2. Study the role of PP4 in IGF-1 signaling and breast tumorigenesis. We will test the hypothesis that PP4 mediates the antagonistic effect of TNF-alpha on IGF-1-stimulated breast cancer cell growth, survival or motility, and thus, acts as a tumor suppressor using gene knockout, siRNA, and dominant-negative mutant approaches. These studies will provide new insight into the novel regulation of IGF-1 signaling by PP4 in breast cancer. Furthermore, this study may lead to the identification of PP4 as a novel target for breast cancer therapeutics.